Yeast growth and library screen.
Standard yeast media and growth conditions were used. The yeast deletion library used is a collection of 4,757 homozygous diploid S. cerevisae strains (BY4743: MATa/MATá his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 lys2Δ0/+ met15Δ0/+ ura3Δ0/ura3Δ0) in which each strain has a single open reading frame replaced with the KanMX4 module. We used homozygous diploid strain library for the original screen because haploid strain appears to have a relatively higher rate of screening background, presumably due to spontaneous mutation. An aliquot of the pooled yeast library was plated on YPG (2% glycerol as carbon source) or YPE (3% ethanol as carbon source) agar plates supplemented with 3–5 μM artemisinin. (Altogether about 100,000 total colonies were plated). Artemisinin-resistant colonies were isolated, and serial dilutions were made and spotted on artemisinin plates to confirm the original phenotype. About a dozen relatively more resistant colonies were chosen, and the corresponding genes were identified by bubble PCR or inverse PCR and DNA sequencing analysis. Phenotypes of these mutants were further confirmed in haploid background (BY4742: MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0).
Artemisinin sensitivity/resistance assay.
For growth in liquid, yeast were grown overnight in YPD medium, spun down, washed three times with YPG medium, and diluted to an A600 of 0.5. Then, 10 μl was inoculated into YPD or YPG media with or without artemisinin, and A600 was measured over time. For growth on agar plates, yeast previously grown in liquid YPD was spotted with 10-fold serial dilutions.
Besides SIP5, genes that may be involved in carbon-source utilization were individually checked for artemisinin sensitivity/resistance. These include GAL83, SIP1, −2, −3, and −4, and SNF1 and −4. snf1 cannot grow on YPG. sip1Δ, sip3Δ, sip4Δ, and gal83Δ display artemisinin resistance.
Mutants of genes in the electron transport chain, including SDH1, −2, −3, and −4, COR1, CYT1, QCR2, −6, −7, −8, −9, and -10, RIP1, INH1, STF1 and -2, and COX4, −5A, −5B, −6, −7, −8, −9, −12, and -13 were individually examined for artemisinin resistance. Among these, SDH1 and -3, CYT1, QCR9, and COX4, −6, −7, and -13 are required for yeast growth on YPG media, precluding analysis of their involvement in artemisinin's action.
Gene deletion and expression.
Gene replacement was achieved by homologous recombination with URA3 or LEU2 as the replacement marker in strain BY4742 background. Gene deletions were verified by PCR. For expression, SIP5, NDI1, NDE1, and PfNDI1 were PCR amplified from BY4743 genomic DNA or a cDNA library of P. falciparum. Amplified target DNAs were then cloned into an ADH1-driven expression vector (pADH1-YES2, a vector modified from pYES2 from Invitrogen (Carlsbad, California, United States), in which the GAL1 promoter was replaced with an ADH1 promoter). Yeast transformation was done by the lithium acetate method. Transformed cells were selected on SD-Ura. Sequences for PCR primers used for SIP5, NDI1, NDE1, and PfNDI1 are available by request.
Analysis of ROS production.
Flow cytometric analysis was used to assay the production of free intracellular radicals. Briefly, cells were incubated with dihydrorhodamine 123 for 2 h and then analyzed by a FACS Calibur (Becton Dickinson, San Jose, California, United States) at a low flow rate with excitation and emission settings of 488 and 525–550 nm (filter FL1), respectively.
Assay of the electrochemical potential.
After treatment in 20 mM HEPES buffer (pH 7.4) containing 50 mM glucose, 1 ml of the cell suspension was incubated with 2 μM Rh123 (rhodamine 123) for 30 min, washed, and then resuspended in 100 μl PBS. Mitochondrial electrochemical potential was expressed as the fluorescence intensity of Rh123, which was read through a FACS Calibur (Becton Dickinson) with excitation at 480 nm and emission at 530 nm.